Background: AKT kinases mediate insulin signaling downstream of phosphatidylinositol-3 kinase (PI3K). Results: AKT binds Sirt2 in insulin-responsive cells and Sirt2 inhibition blocks AKT activation, whereas Sirt2 overexpression sensitizes cells to insulin. Conclusion: Sirt2 deacetylase is an essential factor in AKT activation. Significance: Sirt2 modulators could be useful in treatment of diseases involving AKT, such as type 2 diabetes and cancer.

Cas , which required p130 Cas hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130Cas pathway by expression of a dominant-negative form of p130Cas or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1⅐FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors.

1 AbstractThe specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH 2 -terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the G α13 and Cdo-mediated pathway, in vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development.Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa.

Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at the membrane and the Notch4-intracellular domain (ICD), translocates to the nucleus and regulates gene transcription. Little is known on the mechanisms regulating Notch4-ICD and its nuclear localization. Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that AKT binds Notch4-ICD and phosphorylates all four sites in vitro and in vivo. The phosphorylation in cells is regulated by growth factors and is sensitive to phosphatidyl inositol-3 kinase (PI3K) inhibitors. This phosphorylation generates binding sites to the 14-3-3 regulatory proteins, which are involved in the regulation of nucleocytoplasmic shuttling of target proteins, restricting phosphorylated Notch4-ICD to the cytoplasm. Our findings provide a novel mechanism for Notch4-ICD regulation, suggesting a negative regulatory role for the PI3K-AKT pathway in Notch4 nuclear signaling.

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We previously reported that the level of c-Jun NH 2 -terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, increases dramatically during nerve growth factor (NGF)-induced differentiation of PC12h cells. In the present study, we investigated the function of JSAP1 during PC12h cell differentiation by knocking down the level of JSAP1. The depletion of JSAP1 caused NGF-treated PC12h cells to form aggregates and impaired their differentiation. The aggregation was not observed in JSAP1-depleted cells that were untreated or treated with epidermal growth factor. Immunocytochemical studies indicated that N-cadherin, but not E-cadherin, was localized to sites of cell-cell contact in the aggregated cells. Furthermore, an inhibitory anti-N-cadherin antibody completely blocked the aggregation. Taken together, these results suggest that JSAP1 regulates cell-cell interactions in PC12h cells specifically in the NGF-induced signaling pathway, and does so by modulating N-cadherin. Ó 2006 Elsevier Inc. All rights reserved.Keywords: Cadherin; Differentiation; JNK; MAP kinase; NGF; RNA interference; Scaffold protein Mammalian mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase (JNK), and p38 subgroups, are activated in response to a variety of extracellular and intracellular stimuli [1,2]. Mammalian MAPK signaling pathways, in which MAPK is activated by a phosphorylation relay, from MAPK kinase kinase to MAPK kinase to MAPK, play pivotal roles in multiple cellular processes, including proliferation, differentiation, apoptosis, and migration [1][2][3][4]. Scaffold proteins of the mammalian MAPK cascades are thought to function in the spatio-temporal regulation of these pathways by organizing the MAPK signaling components into functional modules [5,6]. The functional complexes enable the efficient signal transduction of specific MAPK cascades. JNK/ stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3) was initially identified as a JNK-binding protein by our group and others, and biochemical studies have suggested that JSAP1 functions as a scaffold protein for JNK MAPK cascades [7][8][9]. Targeted gene-disruption studies in mice and an in vitro differentiation study with jsap1-deficient mouse ES cells showed that JSAP1 is required for embryonic neurogenesis [10][11][12].The expression level of JSAP1 in rat pheochromocytoma PC12 cells increases dramatically during their nerve growth factor (NGF)-induced differentiation into neuronlike cells [8,13]. However, the physiological function of JSAP1 remains to be elucidated. In the present study, we established PC12 cell lines expressing a short hairpin RNA (shRNA) against JSAP1, in which JSAP1 expression is severely decreased, and we used these JSAP1 knockdown

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Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH 2 -terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of fulllength shRNA-resistant mouse JLP rescued the impaired cell migration of the JLPdepleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein G a13 , showed little or no effect on the cell migration defect. Furthermore, although a constitutively active G a13 enhanced the migration of control HeLa cells, the G a13 -induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with G a13 .Key words: cell migration, MAP kinase, p38, RNA interference, scaffold protein.Abbreviations: DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid; GST, glutathione S-transferase; HEK, human embryonic kidney; JLP, JNK-associated leucine zipper protein; JNK, c-Jun NH2-terminal kinase; JSAP1, JNK/stress-activated protein kinase-associated protein 1; KD, knockdown; MAPK, MAP kinase; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; shRNA, short hairpin RNA.MAP kinase (MAPK) cascades transmit signals through the sequential phosphorylation and activation of threetier kinase modules, consisting of MAPK kinase kinase, MAPK kinase and MAPK. Mammalian MAPK signalling pathways play a key role in multiple cellular functions, such as proliferation, migration and apoptosis (1, 2). The specificity of MAPK signalling is mediated, at least in part, by scaffold proteins. Scaffold proteins for MAPK cascades organize the MAPK signalling components into functional modules, thereby enabling the efficient activation of specific MAPK pathways (3-5). The scaffolding complexes also protect the signalling components within the relevant MAPK modules from undesired activation by other signalling molecules in cells.c-Jun NH 2 -terminal kinase (JNK)-associated leucine zipper protein (JLP) was originally identified as a binding protein for the transcription factor Max, and further biochemical study indicated that JLP functions as a scaffold protein for the JNK and p38 MAPK signalling modules (6). JLP is broadly expressed, and is structurally related to JNK/stress-activated protein kinase-associated protein 1 (JSAP1 or JNK-interacting protein 3), a scaffold protein for JNK cascades (7-9).Studies of JLP have mainly focused on identifying its interacting proteins, which include kinesin light chain 1 and G a13 , the a-subunit of the heteromeric G 13 protein (10,11). JLP has also been reported to play an important role in myogenesis by interacting with the cell-surface p...